Histologic Characteristics and KIT Staining Patterns of Equine Cutaneous Mast Cell Tumors

Abstract

Mast cell tumors are uncommon in horses and typically have a benign clinical course, but there are occasional reports of more aggressive behavior. The objective of this study was to review histologic features and KIT expression patterns of 72 previously diagnosed equine cutaneous mast cell tumors to determine if either is associated with clinical outcomes. Biopsy specimens were reviewed using histologic criteria derived from grading schemes, and KIT antibody expression patterns used in canine tumors and surveys were sent to referring veterinarians for follow-up clinical data. Arabians were overrepresented relative to the reference population. Most tumors were well differentiated with low mitotic rates (96%), and aberrant KIT staining patterns, as described in dogs, were uncommonly identified (12%). Associated clinical disease was uncommon and no tumors exhibited malignant behavior. Overall, KIT staining pattern and histologic features were not associated with poor clinical outcome or abnormal tumor behavior.

Mast cell tumors (MCTs) are relatively rare in horses, comprising 2% to 7% of cutaneous and mucocutaneous tumors in 2 surveys. ^5,6 Most lesions are considered benign, but associated disease has been described, including circulating eosinophilia, hyperfibrinogenemia, pruritis, lymph node involvement, and joint pain resulting in lameness. ^2,4,6 Currently, there are no reliable diagnostic tools available to predict the biologic behavior of these lesions in horses. In dogs, 2 histologic grading schemes are commonly employed for canine cutaneous MCTs and have shown that less differentiated tumors are associated with more aggressive behavior and decreased survival time. ^1,3 Likewise, in dogs, the proto-oncogene c-KIT has been implicated as an important prognostic factor in canine cutaneous MCTs; ^8,9 tumors with c-KIT mutations have been associated with poor clinical outcomes, ^8,10 and aberrant KIT expression has been associated with more aggressive tumor behavior. ⁸ Atypical KIT expression has been identified in 2 separate equine MCT case reports, ^2,7 suggesting that KIT expression may be a valuable prognostic tool for equine tumors. The objective of this study was to retrospectively review a series of equine cutaneous MCTs with respect to histologic features used in canine grading schemes and KIT expression patterns to determine the variability between lesions and any association with clinical outcomes.

In a previous retrospective review of equine skin tumors, 92 MCTs were diagnosed at the Colorado State University (CSU) Veterinary Diagnostic Laboratory between 2000 and 2010⁵ by an American College of Veterinary Pathologists specialist; from those cases, a subset in which tissue was available for immunohistochemistry (IHC) was included in this study. The histologic sections, pathology reports, and clinical notes provided by the submitting veterinarian were all reviewed by a single author (L. Clarke) and the majority of histologic sections and IHC reviewed by multiple authors (L. Clarke, C. Duncan, E. J. Ehrhart, and B. Powers). Histologic characteristics evaluated included demarcation of the lesion, the estimated percentage of mast cells in the lesion, mast cell pattern, cell morphology, granularity, differentiation, mitotic figures, and completeness of excision. For each case, a single slide was selected for IHC based on the quantity and quality of the paraffin-embedded tissues. A standard manual indirect horseradish peroxidase (HRP)–3,3′-diaminobenzidine immunohistochemical technique was used, including heat-induced epitope retrieval (EDTA buffer, pH 9.0; Dako, Carpinteria, CA), endogenous peroxidase blocking, and hematoxylin counterstain. The IHC protocol used 1:500 primary polyclonal rabbit anti–human KIT antibody (A4502; Dako) and prediluted HRP-conjugated secondary antibody (K4061; Dako). Cells of Cajal in equine duodenum and normal mast cells in equine skin were used as the positive control; negative controls were slides with antibody diluent replacing primary antibody. Tumors were classified into 1 of 3 groups according to KIT staining patterns described in the dog. ⁸ A clinical follow-up survey was faxed to the submitting veterinarian requesting information, including patient signalment, size, number, and anatomic location of the lesions, associated clinical disease, abnormal complete blood count (CBC) findings, completeness of surgical excision, postoperative healing, adjunctive therapy, whether there was local recurrence or other mast cell tumors on the horse following the primary excision, and status of the horse at the time of last checkup. Descriptive and comparative statistics were performed using commercially available software (SPSS, Inc, an IBM Company, Chicago, IL).

A total of 72 cutaneous MCTs from 72 horses were included in the study. Basic signalment data were available from the submission forms for 86% (n = 62) of horses. Arabians and Quarter Horses were the most commonly affected breeds (35%, n = 25, and 29%, n = 21, respectively), Paints and Appaloosas each comprised 5% (n = 4) of the cases, and the remaining tumors were from a variety of breeds comprising less than 5% each. From the previously reported equine skin tumors diagnosed at CSU, ⁵ the frequency of MCTs in Arabians was compared with MCTs in all other breeds; the odds of an Arabian being diagnosed with an MCT was 5.1 times (95% confidence interval, 3.4–7.6) greater than all other breeds in this tumor-bearing population. There was no significant association between MCTs and Quarter Horses (P = .20), which were one of the most commonly biopsied breeds in the earlier study. ⁵ Males represented slightly more than half the cases (55%, n = 40) where sex was reported, and age ranged from 3 to 30 years with a mean age of 11 years. Lesions were broadly distributed across the body but most commonly reported on the head (35%, n = 25), limbs (21%, n = 15), and thorax (15%, n = 11). Of the horses with multiple masses reported, 5 horses had multiple MCTs, and 1 each had concurrent sarcoid, eosinophilic granulomas, sebaceous adenoma, collagenolytic granuloma, melanomas, and squamous cell carcinoma. Information regarding the length of time the MCT had been present ranged from 10 days to 3 years. Lesion size ranged from 3 mm to more than 12 cm, with a mean size of 6 cm. Rapid growth or sudden changes in size were noted in 19% (n = 14) of the cases, and 21% (n = 15) were grossly described as ulcerated or draining.

Overall, the lesions were histologically very similar, consisting of multifocal to coalescing aggregates of well-differentiated mast cells with a variable number of eosinophils. In the majority of cases (58.3%, n = 42), there were aggregates of mast cells invading into adjacent tissue, whereas tumors were clearly demarcated in 33.3% (n = 24) of cases. Infrequently (8.3%, n = 6), mast cells were more individualized. Most (44%, n = 32) lesions had less than 25% mast cells, 26% (n = 19) were 25% to 50% mast cells, 18% (n = 13) were 50% to 75% mast cells, and relatively few had more than or equal to 75% mast cells (11%, n = 8). The remaining stroma was primarily collagen and reactive fibroplasia with areas of dystrophic mineralization and often large aggregates of eosinophils. Within the tumor, prevailing patterns seen were mast cells either aggregated into nests of usually greater than 50 cells (53%, n = 38) or forming ribbons of cells (42%, n = 30), but often masses had a mix of these 2 patterns. Excision was complete in 28% (n = 20) of the cases and incomplete in 46% (n = 33), and 26% (n = 19) of the samples were incisional biopsy specimens in which margins could not be evaluated.

Individual mast cells were round to polygonal, with a single tumor having more spindle-shaped cells. In most cases, cells were well differentiated, although the prominence of the granules was variable, with 42% (n = 30) having only faint, homogenously distributed granules and 42% (n = 30) with moderate and 16% (n = 12) with prominent granules. Mitotic figures were uncommon, with only 3 horses (4%) having more than two in ten 400× fields; all of these tumors were well differentiated and well granulated, had minimal anisocytosis and anisokaryosis, and had no other unique histologic features. The majority (88%, n = 63) of tumors were described as having a pattern I (perimembranous) KIT staining pattern. Only 6 (8%) tumors were described as a pattern II (focal perinuclear), and 3 (4%) were described as a pattern III (diffuse cytoplasmic). Of the 9 horses that had KIT scores of II or III, 3 were Arabians, 5 were Quarter Horses, and 1 was a Thoroughbred. All were geldings except for 2 mares (the Thoroughbred and a Quarter Horse), and if age was specified, it ranged from 5 to 25 years. These masses were reported on the face (n = 4), thorax (n = 3), and unspecified (n = 2). There was no significant association (P > .05) between KIT staining pattern and tumor demarcation from adjacent tissue, percentage of mast cells in tumors, cellular morphology, granulation, differentiation, or mitotic figures.

Clinical follow-up information from the veterinarian survey responses was available for 43% (n = 31) of cases. Adjuvant therapy was uncommon and variable; 1 horse received oral cimetidine and intralesional dexamethasone, 1 received cryotherapy, 1 received hyperthermia, and 1 was “injected” with an unknown substance. Seven horses with completely excised MCTs were reported to develop other masses after surgery, either at the surgical site or at other anatomic locations, but none known to be confirmed as MCTs. Gastric ulcers were noted in only 1 case, and this horse had a history of excessive nonsteroidal anti-inflammatory drug use for a case of acute laminitis 3 years previous to the MCT diagnosis. This gelding was also reported to have incomplete healing of the incision site 6 months postoperatively, had additional masses that appeared after the primary surgery, and had been diagnosed with Cushing disease. It is unknown if any of these conditions were related to his preexisting laminitis episode or if the Cushing disease was a complicating factor in his incomplete healing or mass recurrence. Two horses developed subcutaneous edema, both of which had masses in the thoracic region. Peripheral mastocytosis was not reported in any of the horses from which survey information and CBC data were available.

Of the 9 horses with KIT scores of II or III, follow-up data were available for 5; 3 were Quarter Horse geldings, one of which developed edema at the surgical site and the other had additional masses that were never diagnosed. The remaining 2 horses with abnormal KIT patterns were Arabian geldings, neither of which had any clinical complications after mass removal. One Arabian and 1 Quarter Horse had masses that were grossly described as ulcerative in the initial submission report. Of these 5 horses, 2 had masses on the face, 2 had masses in the thoracic region, and 1 was unspecified. Clinical data were available on only 1 of the 3 horses that had masses with a higher mitotic rate, which was reported to not have any related complications during the 4 years after mass removal. This horse was also treated with cryotherapy. Eleven cases (35%) were lost to follow-up, 9 (29%) were still alive, 8 (26%) had died or were euthanized for conditions unrelated to MCT, and 3 (10%) had unknown outcomes. None died or were euthanized for conditions related to MCT.

This study represents the largest set of equine mast cell tumors reviewed to date. Results are similar to previous surveys that had identified a predilection in males, a wide age range, and most MCTs on the head, neck, and limbs. ⁶ No breed predilection had been observed previously, ⁶ but in the present study, Arabians appeared to be overrepresented. The overwhelming majority of MCTs in this study were well differentiated and expressed KIT pattern I. With the exception of the few horses that reportedly developed mild concurrent or postoperative complications, most horses in this study from which clinical history was available recovered without incident. While KIT staining has shown to be prognostic in canine MCTs, ⁸ the results of the present study failed to show a similar association in horses. Given that only 12.5% of cases in the present series were classified as a pattern II or III and clinical follow-up was incomplete, it is likely that the present study lacked sufficient power to detect an association between more aggressive behavior and abnormal KIT staining patterns if one truly exists. In 2 previous equine MCT case reports, both horses had abnormal KIT staining patterns that were described as focal perinuclear (pattern II); 1 horse was reported to have an uncomplicated clinical recovery, ⁷ whereas the other had more severe clinical disease, including edema, multiple masses, and draining tracts. ² Polymerase chain reaction (PCR) for the c-KIT gene has been described as a method for analyzing mutations and, like aberrant expression patterns, has been associated with cellular proliferation and malignant disease in canine MCTs. ¹⁰ Whether these mutations are the cause of aberrant KIT expression is unknown. It is possible that malignant and aggressive behavior may be more closely related to a mutation than the resulting expression pattern; thus, PCR for the c-KIT gene should be investigated as a method for predicting aggressive behavior in equine MCTs as well.

There is debate over whether MCTs in horses are a truly neoplastic process, given their benign clinical and biological behavior, or if they represent a nonneoplastic syndrome similar to uriticaria pigmentosa in humans. ² The prevalence of nonaberrant KIT staining patterns in a large study such as this may be further support for this theory. Other differentials, such as eosinophilic granulomas and cutaneous mastocytosis, should be considered in these cases, and a reevaluation of cellular criteria may be necessary to categorize these masses. KIT staining patterns may also be useful in further characterizing the neoplastic nature of MCTs. Overall, the results of this study show that equine MCTs have a predominantly benign clinical course and that further investigation should be done to identify an accurate prediction method of aggressive behavior.